Detection of selected antimicrobial residues in red meat and kidney of beef cattle slaughtered at Nekemte municipal abattoir, Ethiopia

Abstract Background Antibiotics are commonly used drugs in farm animals for therapeutic, prophylactic and diagnosis purposes. As a result of the use of antibiotics in livestock, residues of antibiotics may be present in animal‐derived foods, especially in meat. This study aimed at determining the antibiotic residues in cattle slaughtered at Nekemte municipal abattoir and assessing the knowledge, attitude and practice (KAP) level of the community about antibiotic residue in food of cattle origin. Materials and methods A cross‐sectional study was conducted on randomly selected kidney and muscle samples slaughtered at Nekemte municipal abattoir. The samples were collected aseptically and analysed using liquid chromatography mass spectrometer. In addition the KAP of cattle handlers, butcher men and meat users were collected using structured questionnaire. The data was analysed by SPSS and intercooled Stata version 7.0, 2001. Results Results are presented as percentages and frequency distributions in tabular and graphical form. From 120 individuals interviewed, only 25.83% knew about withdrawal time and had limited knowledge on effect of antibiotic residue on human health, whereas about 47.5% did not heard about antibiotic residue in meat. Tetracycline was detected in all the sampled meat 60 (100%), whereas oxytetracycline residues were detected in half 15 (50%) of the samples. In the current study doxycycline, sulphadiazine, penicillin G and enrofloxacin were not detected in all samples. Oxytetracycline residue levels ranged from 0.00 to 463.35 µg/kg for the kidney and 0.00 to 354.55 µg/kg for muscle samples. About 10% of kidney and 3.33% muscle samples collected had oxytetracycline residues above maximum residue limits. Conclusion In general, the study revealed that oxytetracycline residues were prevalent among antimicrobial residues analysed from the study area. The study indicated the presence of high antimicrobial residue and hence exposes for antimicrobial resistance of pathogens warranting coordinated effort to mitigate its health effect on the animal and hence human being.


INTRODUCTION
Food safety is of great importance to consumers' health.Currently, hundreds of substances are used as growth enhancers, prophylaxis and management of diseases in food-producing animals (Shahbazi et al., 2015).Antibiotics are among the most widely used drugs.Previous studies have confirmed the inappropriate use of these drugs in animals by livestock owners and pastoralists (Alhaji & Ishola, 2018).The overuse of antibiotics in animals has been connected to the build-up of antibiotic residues in foods derived from animals used for human consumption as well as to the selection pressure for bacteria resistant to antibiotics in both people and animals.The overuse of antibiotics in animals leaves residues in their meat, milk and eggs that eventually find their way into human bodies through bioaccumulation.These residues can be carcinogenic, mutagenic, teratogenic, nephrotoxic, hepatotoxic, bone marrow toxicity or allergic reaction (Nisha, 2008).
Even if there is little data on the true number of people or animals that use drugs irrationally, government authorities' responses to the issue do not seem credible.Study conducted by Melku et al. (2021) in Ethiopia indicates that about half of the drugs are used indiscriminately.Furthermore, there is a lack of awareness and preparedness in dealing with the risk of indiscriminate use of antimicrobials.Food animals slaughtered for domestic and export purposes in the country are not screened for the presence of residues in any of the slaughterhouses.Few studies conducted in Ethiopia provide the information on the level of antibiotics residues (Bedada & Zewde, 2012) where misuse of antibiotics is practiced.
The objective of this study was to determine antibiotic residue level in beef and assess the awareness of the community and other stakeholders about beef antimicrobial residues in Nekemte town.It looked at laying the groundwork for future chances to find out more about national antimicrobial stewardship programmes and research.
The data is helpful in developing a strategic rule aimed at reducing the amount of antibiotic residue found in cattle meat.The results of this investigation advance our understanding of antibiotic residue in food derived from animals.The Ministry of Health, the food industry and other organisations that wish to create regulatory programmes for Ethiopia's food industry will find the knowledge beneficial.

Study area and animals
This study was conducted in Nekemte, East Wollega zone of Oro-

Study populations
The animals were usually brought to Nekemte from Uke, Getema, Diga, Arjo and Sasiga markets and then purchased by restaurant owners and taken to Nekemte town municipal abattoir for slaughter.The animals are slaughtered after ante mortem examination within 1-6 h of their arrival to the slaughterhouse.

Study design
A cross-sectional study was under taken in Nekemte slaughterhouses from October 2021 to November 2022.The cattle come from the surrounding district markets.In this slaughterhouse, an average of 30 heads of cattle are slaughtered daily, three times a week.The study population consisted of apparently healthy slaughtered cattle at slaughterhouses.
On each sampling day 2-3 animals were randomly selected.Kidney and muscle samples were collected from each animal in separate sterile sample containers.

Sample size determination
A total of 60 raw beef samples were randomly collected from Nekemte municipal abattoir found in Nekemte town.A purposive sampling was used to select this slaughterhouse, then random sampling was used to pick the beef samples from slaughterhouse, 30 kidney and 30 muscle samples from slaughterhouse.

Sampling method
On each sampling day, 2-3 randomly selected cattle were selected, and their muscle and kidney were used for further processing.A total of 60 samples 30 each from muscle and kidney) were taken randomly from a slaughterhouse immediately after slaughtering.Samples were packed in polyethylene bags, sealed, labelled and kept in dry ice box stored in −20 • C.

Knowledge, attitude and practice
A total of 120 participants were included in the survey.Purposive sampling technique was used to gather information using pretested questionnaires.The purpose of the questionnaire was to collect interviews using questionnaires targeting 100 respondents living in Nekemte.The number of respondents was determined using the following formula: n = 0.25/SE 2 ; where n is the sample size and SE is the standard error (Arsham, 2002).Considering the non-response rate, 20% of the sample size was added to give a total of 120 participants.
The non-response rate was determined using the formula: n/1 − f,

Determination of antibiotic residue levels
The antibiotic residue level was performed by using high performance liquid chromatography coupled with triple-quadrupole mass spectrometer (LC-MS/MS).In this particular study, HPLC of an Agilent 1290 Infinity II system (Agilent Technologies Ltd.) interfaced to an Agilent 6470 LC/TQ/ triple-quadruple mass spectrometer (Freitas & Ramos, 2014).The HPLC-MS/MS equipped with an Agilent jet stream electrospray ionization source, which was operated in positive mode (AJS-ESI+) and controlled by Mass Hunter software (Doyuk & Dost, 2023).
The antibiotics separation was chromatographically using on Phenomenex Synergi hydro-RP, (4.6 mm × 150 mm; 4 µm, 80 Å dimensions) column with a guard cartridge system (4 × 3.0 mm 2 ).The mobile phase was a binary gradient mobile phase with a flow rate set at 1.0 mL/min for a total run time of 17 min (Table 1).Methanol with 0.1% FA (Mobile phase-A) and acetonitrile with 0.1% (v/v) (Mobile phase-B) were used.

Sample pretreatment
About 500 g of beef muscle tissue (Codex Alimentarius Commission [CAC], 2011) samples and in parallel kidney, samples of the same animals slaughtered at Nekemte town were sampled.Each sample was collected using a sterile sample collection polyethylene/plastic bag individually identified and properly labelled using labelling tape.
Specific numbers, dates, and butcher's shops for the sample collection were recorded.Individually collected and packed samples were placed in an icebox during shipping to animal products and inputs testing centre of Ethiopian Agriculture Authority, and the cold chain was maintained after arrival at the laboratory center.
After removing fats, the samples were minced and homogenized using meat blender.From each homogenized sample, 4.0 g was accurately weighed, in duplicates, in a 50 mL falcon tubes and kept frozen (≤20 • C) until the time of samples extraction and clean up.Before analysis, samples were allowed to defrost at room temperature.

Sample clean up by solid phase extraction
To Then the eluted solutions were directly collected into 15 mL scaled conical plastic centrifuged tubes, which were preplaced in the manifold.
About 5 mL of the clean extracts were collected into another sample tubes and evaporated at 40 • C under a gentle stream of nitrogen gas nearly to dryness (0.10 mL) using MultiVap 54 Lab Tech a nitrogen gas streamed sample concentrator with a 2/3 filled water bath.The sample concentrator was coupled with an online nitrogen gas generator.
Afterwards, the concentrated residues were reconstituted with 1 mL initial mobile phase, recapped and vortexed for 30 s, and centrifuged for 15′ at 4500 rpm at 4 • C. Finally, clear supernatant, supposed to contain antimicrobial residues of interest, was transferred into autosampler vials and closed tightly to make them ready for injection.Finally, a 10 µL injection volume was injected in to the HPLC-MS/MS system.
A given sample was regarded as positive for tetracycline if its retention time and peak corresponded to the standard.Retention time was considered a reasonably unique identifying characteristic of a given analyte.

LC-MS/MS method of analysis (detection and determination methods)
The analysis was performed by UHPLC of an Agilent 1290 Infinity II system (Agilent Technologies Ltd.) interfaced to an Agilent 6470 LC/TQ/ triple-quadrupole mass spectrometer (MS/MS) equipped with an Agilent jet stream electrospray ionization source, which was operated in positive mode (AJS-ESI+) and controlled by MassHunter software.Antimicrobials separation was chromatographically achieved on Phenomenex Synergi hydro-RP (4.6 mm × 150 mm; 4 µm, 80 Å dimensions) column with a guard cartridge system (4 × 3.0 mm 2 ).The mobile phase was a binary gradient mobile phase with a flow rate, which was set at 1.0 mL/min for a total run time of 17 min (Table 1).Methanol with 0.1% FA (mobile phase-A) and acetonitrile with 0.1% (v/v) (mobile phase-B) were used.

Data analysis
The obtained data was captured in Excel and imported into SPSS version 20 software, descriptive and inferential statistics (t test) were used to analysis data.Categorical variables were expressed by proportions and their significance was assessed, when appropriate using chi-square (χ 2 ) test, and a p-value of ≤0.05 was considered statistically significant.
Continuous variables were expressed by means and standard deviations and assessed for statistical significance using the Kruskal-Wallis chi-square test.

RESULTS
The KAP of the cattle handlers regarding antibiotics residue in dairy and beef product was evaluated.
A majority of respondents (70.8%) were in the 20-40 age range, whereas 29.2% were older than 41 years.Male respondents made up 66.7% of the sample, whereas female respondents made up 33.3%.Protestants made up (57.5%) of the respondents followed by orthodox (31.7%) and Muslims (10.8%).In terms of respondents' knowledge of antimicrobial residue, there is a significant difference between those university a degree and those with lower education level p < 0.05 (p = 0.00), but there is no significant difference in respondents' knowledge based on age or sex (p = 0.88) (Table 2).

Respondent's knowledge on antimicrobial use and drug residues
Most of the respondents (52.50%) know drug residues.About 54.17% of the respondents knew the effects of residues in human health.The majority (56.67%) were aware of that economic impact of antibiotic residues.About 74% of the respondents know about drug withdrawal period, whereas 25.8% do not know about withdrawal period and its health effects.Only 40% of respondents know methods to control and prevent drug residues in beef meat, whereas 60% do not.Knowledge on antimicrobial drugs increased as the education level increased.The differences were strongly significant p < 0.05 (Table 3).

Respondent's attitudes on antimicrobial use and drug residues
Approximately 58.33% of participants disagreed that antibiotics used to treat animals end up in animal tissue.About 52.5% of respondents agreed that eating meat contaminated with antibiotics has an adverse effect on one's health.The majority of respondents (68.33%) concurred that a veterinarian's post-treatment guidance may lower the likelihood of antibiotic residue.The majority of respondents (60.83%) thought that heating meat could lower the possibility of antibiotic residue (Table 4).

Respondents' practice about beef
Most respondents purchase meat from butcheries (80%), followed by back yard slaughter (16.67%).Most of the respondents (72.50%) purchased meat, whereas others purchase offal's such as liver (15.83%) and kidney (7.50%).Respondents were reply for consuming meat one to two times only per month where cooking was the most common method (86.67%) of preparation (Table 5).

Detection and quantitative analysis by HPLC-MS/MS
Samples collected from beef cattle were analysed by HPLC for quantification of antimicrobial residue.Retention time was considered a TA B L E 3 Respondents' knowledge on antimicrobial use and drug residues (n = 120).reasonably unique identifying characteristic of a given analyte.Figure 1 shows chromatograms (the visual output of the chromatograph) in which x-axis is the retention time and the y-axis is a signal obtained by ultra-violate diode array detector corresponding to the amount of antibiotics existing in the system.The peaks are characteristic of their identity, with a distribution around the mean position (apex of the peak) that is characteristic of the kinetic properties of the chromatographic system.The area inscribed by the peak is proportional to the amount of substance separated in the chromatographic system.To get the con-centration of an antibiotic sample, a reference standard of a known concentration was injected into the HPLC, and the concentration of the sample was extrapolated from the curve peak area.

Knowledge level assessed
The x-axis is the retention time and the y-axis is a signal obtained by UV diode array detector corresponding to the amount of antibiotics existing in the system.The peaks are characteristic of their identity, with a distribution around the mean position (apex of the peak) that is characteristic of the kinetic properties of the chromatographic system.The area inscribed by the peak is proportional to the amount of  substance separated in the chromatographic system.To get the concentration of an antibiotic sample, a reference standard of a known concentration was injected into the HPLC, and the concentration of the sample was extrapolated from the curve peak area (Table 6).

Proportion of tetracycline antibiotic residues
Out of the total 60 meat samples, 15 (50%) and 60 (100%) had detectable oxytetracycline and tetracycline residues, respectively, whereas doxycycline, sulphadiazine, penicillin G and enrofloxacin were not detected.The ranges for tetracycline residue levels from individual organs were 0.12 to 463.35 µg/Kg for kidney and 0.41 to 354.55 µg/Kg for muscle (Table 7).

TA B L E 6
The residue analysis values in beef meat.Abbreviations: LOD, limit of detection; LOQ, limit of quantification; MRL, maximum residue limit; RV, recovery value.

DISCUSSIONS
Veterinary medications are currently widely utilized to treat and prevent illnesses in animals, encourage healthy growth in animals, and increase feed conversion rates.Nevertheless, veterinary medication use in animals raised for food production may leave parent chemical and/or metabolite residues in food items that are toxic to humans (Getahun et al., 2023).The issue of antibiotic residues in meat is a serious problem that is not effectively addressed in low and middle income countries including Ethiopia.The safety of foods of animal sources regarding drug residues receives suboptimal attention in the country and concerns on the public health impact such as antibiotic resistance of bacteria strains in humans and animals are growing.
Antibiotic overuse and unprocessed antibiotic disposal are putting the environment and its people in danger of a major health hazard.The environment is with an abundance of bacteria and its genes that are resistant to many antibiotics; thus, the effective removal of antibiotics must be addressed right away (Apreja et al., 2022).
This study revealed that knowledge and practice regarding antibiotic residues were generally moderate among cattle handlers in the study area.A large proportion of the participants understood that the presence of antibiotics in meat was a result of antibiotic residues in animal meat, and this agrees with the report by Mgonja et al. (2018), which showed 35% of the respondents were aware about antibiotic residue in meat.Two thirds of the participants confirmed waiting a while after administering antibiotics before slaughtering, and more than half of them had limited knowledge of the concept of withdrawal period.This practiced self-prescription and administration without professionals' consultations.Farmers or cattle handlers result to self-medication due to lack of adequate and high cost of veterinary services as reported by Olasoju et al. (2021).This widespread and unrestricted usage of different antibiotics in food animals without adequate diagnosis, prescription and supervision of veterinarians has contributed greatly to the deposition of residues in animal products (Olatoye & Ehinmowo, 2011).Kinsella et al. (2009) reported that the use of antibiotics in animal agriculture is significantly higher than in human medicine, which could result in antibiotic residue and hence antimicrobial resistance.
Study conducted by Muriuki et al. (2001) in Nairobi and surrounding areas reported 45.6% detectable levels for tetracycline.
Overall, these findings suggest that approximately about 3. In the current study, 50% of oxytetracycline and 100% of tetracycline residue were detected.This finding is lower to report by Bedada and Zewde (2012) at Ethiopia who reported 71.3% oxytetracycline residue level.The current study revealed higher value to the report from Iran who indicated 75% residue level (Baghani et al., 2019).Furthermore, it may indicate that the recommended withdrawal time may not have been respected before slaughtering of the animals.In this study, doxycycline, enrofloxacin and penicillin G were not detected in all samples, which are consistent with the findings by Bedada and Zewde (2012).
Furthermore, the current study found much lower concentration of oxytetracycline in the kidney than another finding reported in Addis Ababa, Adama and Bishoftu slaughterhouses, which reported 99.02, 109.35 and 112.53 µg/respectively (Uma & Ashenef, 2023).The variation in the levels of oxytetracycline residues in the tissue samples could be due to exposure of the animals to antibiotics weeks or even days before slaughter, unauthorized use of the antimicrobials, over use of the drug, inadequate knowledge of the farmers and/or failure to apply instructions on the drug label.In addition, this may be due to the unreasonable use of large quantities of drugs without a professional prescription, the relatively cheap intake of antibiotics, and the inappropriate intake of antibiotics.Again, this antibiotic is widely used in these areas of research, probably due to their affordability, accessibility and broad-spectrum effect.In addition, this may be due to lack of awareness and outreach, which may lead to drug abuse and overuse and may result in failure to observe discontinuation periods.This indicates that there is a need to take appropriate action by the concerned bodies to protect the consumer's health.The current study also recommends further research, particularly to assess the factors that increase the risk of antibiotic residues.

Limitation of the study
The cost of analysis is somewhat expensive, and some butcher houses are not willing to give samples for analysis of antibiotic residue.Additionally, only a few antibiotic analysis protocols were found in the country.

CONCLUSIONS AND RECOMMENDATIONS
The present survey contributes to the better understanding of the current status of cattle handlers' levels of knowledge and practice regard- each sample, 10 millilitres of the extraction solution (2 mL Na 2 EDTA-McIlvaine buffer and 8 mL of acidified graphite carbon nitride with 0.1% FA) were added and mixed by vortexing and shaking using a calibrated solvent dispenser in sequence to the falcon tubes.Then the tubes closed tightly and mixed briefly by vortex for 30 s. Subsequently, the sample mixtures were shaken vigorously for 15 min using a wristaction mechanical shaker.After shaking, the 50 mL sample tubes were centrifuged for 15 min at 4500 rpm at 4 • C. Then samples purified by solid phase extraction (SPE) technique using 12 ports SPE vacuum manifold.After carefully mounting the Oasis hydrophilic lipophilic bond (HLB) SPE cartridges on the vacuum manifold, the supernatant was loaded from the 50 mL falcon tube via Oasis HLB cartridges following the proper conditioning, washing, equilibration and eluting steps.
33% and 10% of muscle and kidney samples collected from Nekemte town had oxytetracycline residues above maximum residual limits, which could have potential health implications for consumers.The high prevalence of oxytetracycline residues observed in the current study probably reflects cattle have sold for slaughter whilst under a therapeutic or prophylactic regimen or animals being slaughtered before the end of the withdrawal period(Aiello & Moses, 2010).Slightly lower report exists elsewhere as compared to the current finding.Duong 2005 reported lower residue level of tetracycline (5.52%) from pork samples.Moreover,Patyra et al. (2020) reported higher residue level 63%-93% from samples of liquid manures and spiked pig and poultry excrement.The prevention levels of antibiotics beyond maximum residue limits require combined and coordinated effort among government agencies, veterinarians and livestock producers.As it obtains in high income countries, the avoidance of meat residues in the livestock industry should take an on-farm team effort that begins with the veterinaryclient-patient-relationship (The College of Veterinarians of Ontario[CVO], 2020).The cattle farm owner/manager/herdsman must work with the farm veterinarian to develop treatment protocols that address the judicious use of antibiotics.Once a decision is made to use antibiotics, protocols must then be put in place to guide employees on the safe way to handle this animal to prevent inadvertent meat residues from occurring.Essentially, treated animals should be identified and antibiotic use must be recorded to prevent residues(CVO, 2020).In this study, participants' demography such as age (p < 0.05) and gender (p < 0.05) were significantly associated with knowledge on antibiotic residues.Education was expected to have a positive association with knowledge as reported byEltayb et al. (2012),Pham-Duc et al. (2019) andIbrahim et al. (2020).The poor knowledge observed among the young educated cattle stake holders could have resulted from lack of awareness of the issues of antimicrobial residues, carefree attitude as well as lack of experience compared to the older stake holders who might have gained experience over the years.In addition, female respondents were likely to have poor knowledge of antibiotic residues than their male counterparts and this is not in agreement with the reports ofOlasoju et al. (2021) in which female participants were more likely to have better knowledge than their male counterparts.On the contrary, a previous report byIbrahim et al. (2020) documented male participants were more likely to have better knowledge than female counterparts.
ing antibiotic residues in current study area.The use of antibiotics to bring about improved performance in growth and feed efficiency, to synchronize or control of reproductive cycle, and to improve breeding performance also often leads to harmful residual effects.It may be due to inadequate awareness and insufficient extension activities that can lead to misuse and overuse of the drug, and possibly failure to observe withdrawal periods that necessitate immediate action.The study indicated the presence of high antimicrobial residue and hence exposes for antimicrobial resistance of pathogens warranting coordinated effort to mitigate its health effect on the animal and hence human being.AUTHOR CONTRIBUTIONSMathewos Tafesse and Debela Abdeta proposed the idea, write the proposal, methodology, data analysis and write original draft.Balachew Bacha supervised and validated all procedures during data collection and accomplishes final correction to the work.Debela Abdeta also writes final draft and takes responsibility in manuscript submission process.

TA B L E 2
Demographic characteristics of respondents (n = 120).
Attitudes of the respondent on the antibiotic residue in meat (n = 120).
TA B L E 5